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Abstract

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al., 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells.

Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al., 1990; Ouellette et al., 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Cell pellet: Prepare cell pellets (1 x 106 - 2 x 106) in 2 ml polypropylene screw-cap tubes. After removing the supernatant, cell pellets can be frozen at -80 °C.Note: It is preferred to wash cell pellet with 1x PBS, but this step is not required because the small amount of medium in the cell pellet does not interfere with subsequent steps.

Cell lysis: Re-suspend the cells that are fresh or just thawed on ice from -80 °C with 200 μl 1x PBS and add 20 μl proteinase K (20 mg/ml).

DNA extraction: Use DNeasy Blood and Tissue Kit to get high DNA yield. Quantify DNA samples by Nanodrop.Note: Multiple methods are available to extract DNA from cells. We prefer the commercial kit since it can easily be performed to get high yield DNA.

Add 5 μl loading dye to the samples. While 0.5x TBE can be used to analyze less than ~8 kb, 1x TAE buffer can be used for the ones that have longer telomeres (8 to 20 kb range) to have better separation.

400 ml volume is good enough for 0.7% (w/v) agarose gel in large gel system.

Radiolabeled TRF marker can be visualized after hybridization with telomere sequence-specific probe. The unlabeled, digested plasmid DNA can be visualized with Gel Red, not with the telomere sequence specific probe.

To prevent leaking of the gel from the gel tray: Wait 20 min after agarose is dissolved in microwave. During this time, seal the space between the gel tray and gaskets (edges of gel tray) with 5-10 ml agarose gel (Figure 2A). Wait 30 min following the pour of the gel (do not forget the put comb when you pour the gel).

Higher concentration of agarose in buffer will cause poor separation of samples and TRF marker (Figure 2B) and also processing gel drying will take a much more time than lower concentration of agarose.

Figure 2. Preparation of agarose gel electrophoresis. A. This figure shows how to seal between the gaskets and gel tray with agarose gel to prevent leaking. B. The separation difference between 0.7% and 1.4% agarose gel. While 0.7% agarose gel shows good separation on the TRF ladder, TRF ladder on 1.4% agarose gel is not separated well. 25 μl and 12.5 μl ladder was loaded in each lane of 0.7% and 1.4% gels, respectively. TRF ladder 1 and 2 in 1.4% gel are the same ladders. TRF ladder units are kilobase (kb).

DNA Hybridization:

Denature the gel for 20 min in 1.5 M NaCl and 0.5 M NaOH solution (pH 13.2) in a Pyrex® container (slowly shake). Make sure that denaturing solution covers the gel during shaking.

Rinse gel with MilliQ® water to remove NaOH.

Put the gel upside down on 2 sheets of 3MM Whatman® paper and wrap on the top of the gel (Figure 3A and B).

Figure 3. The method for drying the gel (A and B). Whatman® papers are located at the bottom, gel is located in between Whatman® papers and plastic wrap is on top of the gel.

Dry the gel using a gel dryer (56 °C for ~3 h).

Transfer the gel to a Pyrex® container, rinse with MilliQ® water and remove the Whatman® paper.

Washing: Wash the gel once in 2x SSC, 0.1% SDS solution for 15 min at 42 °C, then wash the gel twice in 0.5x SSC, 0.1% SDS solution for 15 min at 42 °C. Finally, wash the gel twice in 0.5x SSC, 1% SDS for 15 min at 42 °C. 10-15 ml washing solution can be used for each washing step at 42 °C rotating hybridization oven.Note: Prepare the washing solutions in the following order: SSC, water, SDS so they dissolve easily.

Exposure: Prepare the gel for scanning. Briefly, wrap the gel with plastic (Saran type) wrap in the cassette and put the screen on the gel (Figure 4). Expose it at least 4 h, preferably overnight. Scan the screen on Typhoon PhosphorImager.

Figure 4. Illustrates the preparation of the gel for scanning step by step. Prepare the saran plastic wrap in an appropriate cassette size and spread it into the cassette. Then, put gel onto this plastic wrap and cover the gel by wrapping with additional plastic wrap. Place screen on top of the gel that is surrounded plastic wrap, then incubate it in a dark place. All steps should be done behind a protective shield.

Measure the distance for each marker band from the top in Image Quant software (X: measured distance, Y: molecular weight) (Figure 5A).

Draw a rectangle with 150 rows around the samples and background (empty lane) in Image Quant software (Figure 5B).

Get the intensity of each 150 boxes for each sample (Volume reports from analysis tool give the intensity values) in Image Quant software. Export volume values from Image Quant to Excel.

Open Graph Pad Prism and create a new data and table. Check “Y: Enter and plot a single Y value for each point”. First column (X): measured distance, second column (Y): molecular weight. Go to analysis, analyze, XY analyses, nonlinear regression (curve fit) and select one phase exponential decay. Go to tab “Range”: check “create a table of XY coordinates of 150 points that define the curve”. Nonlinear fit of Data1 contains molecular weight for each distance in the Y row.

Calculate the average telomere length using following formula =Σ(Inti)/ Σ(Inti/MWi). Figure 5 C and D show an example for TRF gel and average telomeric lengths for some non-small cell lung cancer cell lines, respectively.

10x PBS buffer-phosphate buffer saline
Powder for 5 L of 10x is ready to use for preparation of 5 L of concentrated 10x phosphate-buffered saline (PBS)
Prepare 5 L milliQ® water and add PBS powder
Next add large stir bar and place on stirrer until solids are dissolved

Some of these protocols were adapted from previously published studies (Herbert et al., 2003). We thank Zeliha Gunnur Dikmen for her help in acquisition of TRAP gel and Abhijit Bugde from the Live Cell Imaging Facility at UT Southwestern for his assistance with the imaging and analysis part of Telomere dysfunction Induced Foci (TIF) analysis.